RANKL-evoked [Ca2+]i oscillations play a switch-on role in osteoclast differentiation through the NFAT2 activation pathway, however, the question remains as to how RANKL evokes the [Ca2+]j oscillation-NFAT2 pathway. Some evidence has shown that cytosolic [Ca2+]i oscillations are generated mainly by influx of extracellular Ca2+ through multiple channels, which include L- and N-type channels, and Ca2+ influx is necessary for maintenance of oscillations. Schiff et al. (Schiff et al., 2000) first reported that RGS12 is capable of direct interaction with the tyrosine-phosphorylated calcium channel in culture of primary dorsal root ganglion neurons through its PTB domain. Recruitment of RGS proteins to G-protein effectors may represent an additional mechanism for signal termination in G-protein-coupled pathways/Using differential screening, we have found that regulator of G-protein signaling 12 gene (RGS12) is predominantly expressed in osteoclast like cells (OCLs). Knockdown of RGS12 expression using RNA interference (RNAi) inhibited the osteoclast differentiation induced by RANKL. The failure of osteoclast differentiation results from the absence of [Ca2+]i oscillations and NFAT2 expression. Our studies further revealed that RGS12 directly interacted with the N type calcium channel to likely regulate [Ca2+]i oscillations and that RGS12 binds with calcium sensing receptor (CaR). These in vitro data suggested that RGS12, the largest member of the RGS protein family and a multi-domain RGS protein, may play roles in numerous signaling regulatory elements. So far, little is known about the in vivo function of RGS12 and the role and mechanism of its multi-domains in osteoclast differentiation and bone resorption. Based on our preliminary data and the cited research, we hypothesize that RGS12 interacts with calcium channels, CaR, G-protein and other heterodimerization partners, to regulate generation of [Ca2+]i oscillations and trigger osteoclast differentiation. To test this hypothesis, we propose the following three Specific Aims: Aim 1. To characterize the role of RGS12 in differentiating osteoclasts and mature osteoclasts by using lentivirus RNAi and overexpression systems. Aim 2. To identify the mechanism of RGS12 action during osteoclast differentiation by characterization of RGS12 heterodimerization partners. Aim 3. To define the role and mechanism of RGS12 in vivo in bone remodeling by osteoclast-specific targeted disruption of the RGS12 gene using Cre/loxP technology!